Rapid flip-flop of phospholipids in endoplasmic reticulum membranes studied by a stopped-flow approach

The transbilayer movement of short-chain spin-labeled and fluorescent 7-nit robenz-2-oxa-1,3-diazol-4-yl (NBD) phospholipid analogs in rat liver micros omes is measured by stopped-flow mixing of labeled microsomes with bovine s erum albumin (BSA) solution. Extraction of analogs from the outer leaflet o f microsomes to BSA can be directly monitored in conjunction with electron paramagnetic resonance or fluorescence spectroscopy by taking advantage of the fact that the signal of spin-labeled or fluorescent analogs bound to BS A is different from that of the analogs inserted into membranes. From the s ignal kinetics, the transbilayer movement and the distribution of analogs i n microsomal membranes can be derived provided the extraction of analogs by BSA is much faster in comparison to the transbilayer movement of analogs. Half-times of the back-exchange for spin-labeled and fluorescent analogs we re <3.5 and <9.5 s, respectively. The unprecedented time resolution of the assay revealed that the transbilayer movement of spin-labeled analogs is mu ch faster than previously reported. The half-time of the movement was about 16 s or even less at room temperature. Transmembrane movement of NBD-label ed analogs was six- to eightfold slower than that of spin-labeled analogs.